Abstract

The aim of this study was to develop and use in vivo bioluminescent imaging to track luciferase-labeled acute myelogenous leukemia (AML) stem cells in immunedeficient mice using AML blood samples collected from patients and subsequently frozen. The ability to utilize frozen pools as viable reservoirs for AML stem cells will allow the freezing back of samples from the clinic for later use as well as the use of previously frozen samples for engraftment and therapeutic studies. Using thawed, isolated cells, we assessed AML stem cell engraftment and quantitatively compared it to fresh AML stem cell engraftment in a xenogeneic transplant model using in vivo bioluminescent imaging. Frozen AML stem cell engraftment was also compared to both fresh and frozen normal human hematopoietic stem cell (HSC) engraftment. Human hematopoietic stem cells (CD34+CD38-) were isolated from both normal frozen cord blood and from peripheral blood of AML patients using modified Miltenyi Biotec (Auburn, CA) magnetic bead isolation kits. Isolated cells were transduced with a lentiviral vector (HIV-luc) pseudotyped with the VSV envelope able to express the firefly luciferase (fluc) gene in human HSC. Using a Caliper Life Sciences/Xenogen IVIS 200 optical imaging system, cells were assayed for fluc expression 48 hours after transduction. Transduced HSC and AML cells (6 × 104 each) were transplanted intravenously into sublethally irradiated adult NOD/SCID/IL2Rγnull mice and mice were imaged serially from day 1 up to day 160 post transplantation. Observed bioluminescent signal and patterns from thawed AML stem cell engraftment are similar to our results obtained using fresh AML stem cells in mice. Luminescent signal from peripheral blood and spleen engraftment of frozen AML stem cells was detectable within one day post-transplantation. Both fresh and frozen AML stem cell populations exhibited diffuse whole body signal, indicating luciferase-labeled stem cells in the peripheral blood, similar to a hallmark of the disease in humans. The presence of blast cells consistent with AML disease was observed by blood smears taken from mice engrafted with frozen AML stem cells. To our knowledge, we have demonstrated for the first time that frozen AML stem cells can be efficiently labeled with a luciferase enzyme for dynamic tracking in vivo. This work has resulted in a useful animal model that will enable us to elucidate the engraftment patterns of AML cells in living animals, learn the dynamics of the interplay of different stem cell populations, and provide information on the efficacy of new treatments in the future.